Antiproliferative activity of antimicrobial peptides and bioactive compounds from the mangrove Glutamicibacter mysorens

The Glutamicibacter group of microbes is known for antibiotic and enzyme production. Antibiotics and enzymes produced by them are important in the control, protection, and treatment of chronic human diseases. In this study, the Glutamicibacter mysorens (G. mysorens) strain MW647910.1 was isolated from mangrove soil in the Mangalore region of India. After optimization of growth conditions for G. mysorens on starch casein agar media, the micromorphology of G. mysorens was found to be spirally coiled spore chain, each spore visualized as an elongated cylindrical hairy appearance with curved edges visualized through Field Emission Scanning Electron Microscopy (FESEM) analysis. The culture phenotype with filamentous mycelia, brown pigmentation, and ash–colored spore production was observed. The intracellular extract of G. mysorens characterized through GCMS analysis detected bioactive compounds reported for pharmacological applications. The majority of bioactive compounds identified in intracellular extract when compared to the NIST library revealed molecular weight ranging below 1kgmole−1. The Sephadex G-10 could result in 10.66 fold purification and eluted peak protein fraction showed significant anticancer activity on the prostate cancer cell line. Liquid Chromatography–Mass Spectrometry (LC–MS) analysis revealed Kinetin-9-ribose and Embinin with a molecular weight below 1 kDa. This study showed small molecular weight bioactive compounds produced from microbial origin possess dual roles, acting as antimicrobial peptides (AMPs) and anticancer peptides (ACPs). Hence, the bioactive compounds produced from microbial origin are a promising source of future therapeutics.


Introduction
The environmental conditions in a particular ecosystem play an important role in determining biodiversity composition. High tides, hypersaline water, significant temperature fluctuations, and optimal flora and fauna diversity are just a few of the distinctive environmental characteristics of the mangrove ecosystem (Karthik et al., 2020). Microbes can better adapt to any extreme environment in these vulnerable situations. The isolation of bioactive chemicals will be greatly aided by this habitat (Alongi, 2015).
Actinomyces word derived from the words "atkis" which means "a ray" and "mykes" which means "fungi" are filamentous, Gram-positive bacteria distinguished by different coloration and spore production at maturity (Chater, 2006). Actinomyces share the characteristics of bacteria and fungi. The Actinomyces group's genetic and environmental flexibility facilitates the development of worthwhile bioactive substances. Actinomyces contribute more in enzyme production to pharmacological industries for the treatment, and prevention of various ailments (Chater, 2013).
The various species of genus Glutamicibacter shown huge biological importance as detailed above. Whereas G. creatinolyticus shown resistance to antibiotics as well as heavy metals (copper, arsenic, cadmium, cobalt, zinc, and chromium;Santos et al., 2020). The G. arilaitensis produced pink colored pigment and coprophorphyrin binds zinc and regulates in cheese rinds (Cleary et al., 2018). Another Gluamicibacter sps. Possessing genes that regulates the growth of plant under saline conditions, cold adaptation, efficient degradation and chitinase enzyme producing genes which help in control the growth of pathogenic bacteria (Borker et al., 2021;Fu et al., 2021). While G. nicotianae involved in heavy metals degradation (Wang et al., 2021). The G. mishrai and halophytocola isolated from Andaman sea sample. Genes involved in cell wall biogenesis, replication, recombination, repair mechanism and amino acid metabolism along possess important role in physiology and behavior of insects Das et al., 2020;.
Antimicrobial peptides (AMPs) are peptides with antimicrobial properties. In multicellular organisms, these positively charged host defense molecules, or AMPs, serve as the initial line of protection. Many AMP's from both prokaryotes and eukaryotes have been categorized (Brandenburg et al., 2012;Desriac et al., 2013). Several genera of AMP's -producing microorganisms have been discovered, including bacteriocins produced by Leuconostoc gelidum, Enterococcus faecium, and other species (Juturu and Wu, 2018;Khodaei and Sh, 2018). Microcins A and B, antimicrobial bacteriocins derived from Streptomyces pluripotens, have been shown to be effective against Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, and Listeria monocytogenes (Collin and Maxwell, 2019;Kurnianto et al., 2021).These AMPs have been found to be effective in the treatment of a broad range of ailments (Sugrue et al., 2019;Karthik et al., 2020;Khadayat et al., 2020;Zhang et al., 2020). AMP's are peptides derived from microbes that exhibit antimicrobial activity. AMPs have been shown to target cell walls or cell membranes, permitting them to penetrate cells and affect vital components while inhibiting growth (Desriac et al., 2013;Wang et al., 2020). As a result of their targetspecific activity against resistant microbial species, AMPs are thought to be anti-microbial compounds.
Peptides with selective action and non-selective activity, i.e., those that have activity against bacteria, cancer cells, and healthy cells, can be categorized as having antitumor activity in Hoskin and Ramamoorthy's investigations (Hoskin and Ramamoorthy, 2008).The peptides have antibacterial and anticancer properties, but not against normal cells. Cecropins, buforins, and magainins, among other peptides, have demonstrated anticancer effects without harming normal eukaryotic cells (Cruciani et al., 1991;Cho et al., 2009). These studies go into great detail and provide a compelling case for the fact that many peptides have biological activity in a variety of dimensions and properties and can possess dual activity as AMPs and ACPs. Therefore, we are searching for mangrove soil Actinomyces in the Mangalore region to isolate and characterize bioactive peptides that can function as both AMPs and ACPs.
In our previous study, we reported the detailed procedures for isolation, microscopic and macroscopic characters, identified as Glutamicibacter mysorens with GenBank accession number MW647910.1, the intracellular protein; extraction, estimation, along with their potential antimicrobial activity was observed against test pathogens Salmonella typhimurium (ATCC23564), Staphylococcus aureus (ATCC6538P),Bacillus cereus (ATCC10876), Proteus vulgaris (ATCC13315), and Pseudomonas aeruginosa (ATCC9027) cultures. The protein was characterized through LCMS and SDS PAGE techniques and small peptides were detected (Karthik and Kalyani, 2021).
In this study, the optimization of suitable growth media for G. mysorens and its micromorphology were analyzed using FESEM. The isolation of intracellular extract of G. mysorens was characterized through GCMS and LCMS. These GCMS studies revealed a large number of small bioactive compounds that possess significant biological activities are discussed. Whereas the LCMS studies resulted in the detection of low molecular weight Kinetin-9-ribose and Embinin showed significant anti-tumor potential against PC3 cell line in comparison to standard cisplatin drug.  (Mohan et al., 2013;Sridevi et al., 2015;Sapkota et al., 2020).

Cultural characteristics
The isolated G. mysorens strain was subjected to FESEM analysis at different objectives distances; spore structure (1 and 2 μm) mycelial structure (10 and 20 μm) to visualize the complete micromorphological structures. The sequencing and identification of G. mysorens are reported by Karthik and Kalyani (2021).

Intracellular extract
The G. mysorens strain was grown in SCN broth for 7 days at 30 ± 2°C with continuous shaking at 100 rpm. Centrifugation at 7000 rpm for 8 min separated the cultured biomass cells, which were then washed twice using phosphate-buffered saline devoid of Mg 2+ and Ca 2+ and centrifuged again. The cells were then re-suspended in 10 ml of chilled acetone for 5 min before being centrifuged at 7,000 rpm for 8 min. The intracellular extract was incubated for 2 min with 1.0 ml of 1% SDS after the traces of acetone was removed with a nitrogen stream (Bhaduri and Demchick, 1983). This intracellular extract was characterized using spectrometric (LCMS, GCMS) tools along with a comparison to the NIST library.

Gas chromatography-mass spectroscopic analysis
The following equipment was assessed for the GC-MS studies of G. mysorens intracellular extract: a PerkinElmer Clarus 680 Gas Chromatograph and a PerkinElmer Clarus SQ 8C Mass Spectrometer. A PerkinElmer Elite-5MS standard column with dimensions of 30 m long x 0.250 mm inner diameter x 1 micron (60-350°C) is utilized in the equipment. With an equivalence ratio of 10:1, the injected volume of 2 μl was completely run for 26.6 min. Helium is used as the carrier gas, with a flow rate of 2 ml/min. The source temperature was set to 230 degrees Celsius, and the inlet temperature was set to 250 degrees Celsius. The oven temperature was initially set to 80°C with a hold time of 2.0 min; ramp1 was set to 10.0 /min to 150°C with a hold time of 1.0 min; and ramp2 was set to 15.0 /min to 250°C with a hold time of 10.0 min. The components were identified by comparing them to those contained in the NIST computer library, which was linked to the GC-MS apparatus, and the results were published.

Gel filtration
The microbial proteins were purified using SephadexG-10. For 5 h, the Sephadex G-10 was allowed to swell in excess of dH 2 O in a boiling water bath. After decanting the gel to remove fines, it was equilibrated with 0.05 M sodium phosphate buffer, pH 7.0. Under gravity, the gel was packed into a 1.0 cm × 110.0 cm column. At a flow rate of 10 ml/h, the column was standardized with two-bed volumes of phosphate buffer of concentration 0.05 M, pH 7.0. The 20 mg of isolate protein sample was loaded onto the gel, eluted with 0.05 M sodium phosphate buffer, pH 7.0, and 2.0 ml fractions were collected and further analyzed (Bharadwaj et al., 2018).

Liquid chromatography-mass spectrophotometer
The Sephadex G-10 peak fraction was analyzed using LC-MS, model Synapt G2, an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with mobile phases A: 0.1% Formic acid in Water and mobile phase B: 0.1% Formic acid in Acetonitrile with the mass analysis capabilities of mass spectrometry (MS) an Agilent 1100 LC system with a vacuum degasser, A BEH C18, 50 mm × 1.0 mm, 1.7 μm C18 column (Waters, United States) was used to achieve chromatographic separation in comparison to the NIST computer library.

MTT assay
Prostate cancer cells (PC3) procured from NCCS Pune; were harvested in T-25 flasks for the in vitro studies. PC3 cells were trypsinized and aspirated into a 5 ml centrifuge tube. After centrifugation at 300 rpm for 10 min, the cell pellet was separated. The cell count was adjusted using DMEM HG medium so that 200 μl of suspension contained approximately 10,000 cells. In an ESCO model CLM170B-8-UV CO 2 incubator, a 200 μl cell suspension was added to each well of the 96-well microtiter plate, and the plate was incubated for 24 h at 37°C and 5% CO 2 atmosphere. After 24 h, the spent medium was aspirated. In each well, 200 μl of various test drug concentrations and the standard drug cisplatin were added. After that, the plates were incubated for 24 h at 37°C and 5% CO 2 . The drug-containing media was aspirated after the plate was removed from the incubator. The plate was then incubated for 3 h at 37\u00B0C and 5% CO 2 atmosphere with 200 μl of medium containing 10% MTT reagent in each well to achieve a final concentration of 0.5 mg/ml. The culture medium was completely removed without disturbing the formed crystals. To solubilize the formed formazan, the plate was gently shaken in a gyrator shaker with 100 μl of solubilization solution (DMSO). The absorbance was read at 570 and 630 nm using the microplate reader of a Multiskan sky ELISA spectrophotometer.

Results and discussion
The Mangrove region in Jeppinamogaru located at Mangalore, India, served as a suitable source for isolating G. mysorens strain. The G. mysorens strain received a GenBank accession number MW647910.1 and was isolated and their biological activities were reported by Karthik and Kalyani (2021). In continuation to previous work; initially, the G. mysorens strain was observed for morphological characteristics after performing FESEM analysis. Also, biologically important chemical components present in the intracellular extract of the G. mysorens strain were characterized using GCMS and a partially purified protein sample Frontiers in Microbiology 04 frontiersin.org was characterized using LCMS and have a shown significant number of bioactive compounds. The cultural characteristics of mangrove adapted G. mysorens strain upon growth on starch casein nitrate agar medium exhibited as white colored filamentous mycelia and at maturity showed ash-colored spores. Production of brown pigmentation on SCNA media was observed. Further microscopic analysis showed Gram staining positive. The isolate when further subjected to FESEM microscopic studies revealed mycelial morphological characteristics of the genus Glutamicibacter. Further, the culture showed filamentous mycelia possessing spirally coiled spore chains. Each spore is visualized as an elongated cylindrical hairy appearance with curved edges as shown in Figure 1. The G. mysorens when grown on different Actinomyces-specific media have shown distinctive phenotypic characteristics as listed in Table 1. Excellent growth was achieved on starch casein nitrate agar, whereas good growth was seen on, glucose leucine agar, yeast extract agar, and nutrient agar media. Moderate growth was seen on sucrose peptone agar, and malt extract agar. Whereas in another study, lysogeny agar was chosen as the best growth media for G. mysorens according to  and Deb et al. (2020).
In our previous report, the G. mysorens strain when subjected to simple and rapid disruption followed according to the method of Bhaduri yielded significant intracellular extraction in buffer (Bhaduri and Demchick, 1983). A 20 mg of protein was loaded on top of the column and 2 ml fractions were collected and about 2.5 times (216 ml) bed volumes of protein elutions were collected. The absorbance of protein fractions was checked at 280 nm and graphs were plotted. The Frontiers in Microbiology 05 frontiersin.org X-axis indicates fraction numbers and absorbance plotted on Y-axis for each fraction collected from Sephadex G-10 column chromatography as showed in Figure 2. This column separation chromatography purifies 10.66 folds as detailed in Table 2. The GCMS studies depicted the presence of 155 bioactive molecules present in the intracellular extract of G. mysorens and the obtained elution profile is as shown in Figure 3. Whereas GCMS analysis depicted the highest probable compounds such as Cyclopentane undecanoic acid, methyl ester 22.7% and Glutaric acid, 2,2-dichloroethyl 3-fluorophenyl ester 34% probability as shown in Figure 4. All the compounds detected through GCMS showed low molecular weight below 1Kgmol −1 with various pharmacological applications. The majority of bioactive compounds have shown antimicrobial, enzyme inhibitors, activators, antioxidants, anti-inflammatory, anticancer, agrochemical, insecticide, anti-obese, and many other applications as listed in Table 3. The intracellular extract of G. mysorens had shown potent antimicrobial activity to a broad spectrum of test pathogens such as Salmonella typhimurium (ATCC23564), Staphylococcus aureus (ATCC6538P), Pseudomonas aeruginosa (ATCC9027), Proteus vulgaris (ATCC13315), and Bacillus cereus (ATCC10876) cultures. In order to focus further on prominent bioactive compounds the intracellular extract was partially purified using a Sephadex G-10 column. The eluted peak fraction upon spectrophotometry and electrophoretic analysis revealed the presence of peptide and is reported in our previous article (Karthik and Kalyani, 2021). A similar study was illustrated on 41 different Actinomyces species and majority isolates shown antagonist activity against Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae (Sapkota et al., 2020). One of the previous study; extracellular protein of Actinomyces are actively producers for enzyme ligno cellulase (Clark Mason et al., 1988). The eluted peak fraction for proteins of G. mysorens has shown significant activity for different concentrations 50 μg of protein fraction showed 24% antiproliferative activity against prostate cancer PC3 cell line, for 100 μg 35% antiproliferative activity was observed, for 150 μg 47% antiproliferative activity was observed and for 200 μg 56% antiproliferative activity was observed in comparison with standard drug cisplatin at 5 μg showed 47% antiproliferative activity as showed in Figure 5.
Similar studies reported that other bioactive compounds isolated from the genus Glutamicibacter have been characterized for antimicrobial activity (Phuong and Diep, 2020;Xiong et al., 2020). In another study reported that plant-growth promoting bioactive Elution profile of Glutamicibacter mysorens by using Sephadex G-10 column chromatography. Elution profile of GCMS analysis for Glutamicibacter mysorens intracellular extract.
Frontiers in Microbiology 06 frontiersin.org compounds was produced by Glutamicibacter halophytocola coastal region of China . Whereas another study describes the anti-fungal efficiency of the Glutamicibacter genus with chitin hydrolyzing activity (Asif et al., 2020). The intracellular protein extraction already reported in our previous studies characterized for an antimicrobial activity that can be considered as antimicrobial peptides (AMPs) from the microbial origin (Karthik and Kalyani, 2021). In the present work the G. mysorens protein fraction is also exhibiting antiproliferative activity against cancerous cells acting also as anticancer peptides (ACP's) and the protein molecules detected and characterized by LC-MS analysis. We are also reporting GCMS analysis and detected bioactive compounds from G. mysorens.
As discussed above the Sephadex G-10 eluted peak protein fraction was further subjected to LCMS analysis. The LCMS analysis and elution profile as shown in Figure 6, revealed the detection of pharmacologically applicable bioactive peptide compounds. With respect to elution peak from LCMS analysis and detection through the NIST, the computer library resulted in the identification of Kinetin-9-riboside and Embinin. The detected Kinetin-9-riboside with 347 Da molecular weight structure and mass confirmation are shown in Figure 7. The mass confirmation and structure of Embinin with a molecular weight of 606 Da showed in Figure 8. These bioactive molecules are well-known for their effective activity in various biological applications.
In a previous study, the therapeutic and biological studies of Kinetin-9-riboside as an immuno-stimulant; immuno-stimulatory activities, and their uses as an adjuvant were reported. Because mutations in induced putative kinase 1 (PINK1) induce severe Parkinson's disease, there's a lot of interest in finding small molecules that boost PINK1's kinase activity. Several studies on the design, synthesis, serum stability and hydrolysis of four kinetin riboside ProTides have been published. These ProTides, in combination with kinetin riboside, activated PINK1 in cells that had not been depolarized by mitochondria. This demonstrates the therapeutic potential of modified nucleosides and their phosphate prodrugs for Parkinson's disease, the second most common neurodegenerative disease (Osgerby et al., 2017). Another study found that the epithelial-mesenchymal transition (EMT) is a molecular phenomenon associated with increased vimentin expression and raised activity of transcriptional factors (Snail, Twist) that inhibit E-cadherin. EMT has been linked to prostate cancer metastatic potential, therapy resistance, and poor outcomes. Kinetin riboside (KR) is a naturally occurring cytokinin with effective anticancer activity against several human cancer cell lines. mRNA and protein levels of AR, E-, N-cadherins, Vimentin, Snail, Twist, and MMPs were measured using Western Blot and RT-PCR or RQ-PCR techniques to determine the effect of KR on human prostate cell lines.KR inhibited the growth of human prostate cancer cells and, to a lesser extent, normal cells. The cell type and androgen sensitivity determined this effect. KR also decreased the level of p-Akt, which is involved in androgen signaling modulation. When cancer cell lines are exposed to KR, the anti-apoptotic Bcl-2 protein is down-regulated, whereas the Bax protein is up-regulated. KR was involved in E-cadherin re-expression as well as pivotal changes in cell migration. Taken together, the findings suggest that, for the first time, KR can be anticipated as a factor for signaling pathway regulation that involves the inhibition of the development of aggressive forms of prostate cancer, potentially leading to future therapeutic interventions. As a result, research indicates that KR is an effective inhibitor of EMT in human prostate cells (Thakor et al., 2016;Dulińska-Litewka et al., 2020).
Whereas Embinin is a C-Glycosyl flavone and has a wide therapeutic applications in cardiovascular diseases (Ivkin et al.,        Elution profile of intracellular protein extract from Glutamicibacter mysorens by LCMS. Frontiers in Microbiology 14 frontiersin.org 2018). Another study reports the production of Embinin frompetals of Iris germanica Linnaeus and Iris lactea Leaves (Kawase and Yagishita, 1968;Chen et al., 2018). Our study elucidates the cytotoxicity activity of G. mysorens bioactive peptide as characterized by LCMS/MS revealed the presence of Kinetin-9-Riboside and Embininin the peptide fraction showing its antiproliferative effect on the prostate cancer cell line. Thus microbial-originated intracellular peptides have potential antimicrobial (AMPs) and anticancer (ACPs) have been significantly substantiated in our studies.

Conclusion
The present study is illustrative for exploring untapped mangrove habitat in the Mangalore region of Karnataka. In our study, we could demonstrate that mangrove G. mysorens is an efficient microbe to produce bioactive compounds and enzymes responsible for both antimicrobial and anticancer activity. The antimicrobial potentiality was detailed in our previous article. In this present study; anticancer activity on prostate cancer cell lines and to treat various other related ailments. Peptides from reliable sources such as Actinomyces could be demonstrated as having dual roles as AMPs as well as ACPs. Hence, this study supports and proves that the genus Glutamicibacter is an effective microbial group for the isolation of peptides to treat multidrugresistant pathogens.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/ repositories and accession number(s) can be found at: NCBI -MW647910.

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.